A high-efficiency separation and purification method of MLnM type medium-long chain triglyceride

Abstract

The application discloses a kind of high-efficiency separation and purification methods of MLnM type medium-long chain triglyceride, belongs to the technical field of separation and purification, comprising the following steps: S1, preparation of crude product;S2, crude product is mixed with adsorbent and carries out static adsorption;S3, static adsorption product is eluted by column chromatography, and MLnM type triglyceride is separated out by dynamic chromatography.The application uses the above-mentioned high-efficiency separation and purification method of MLnM type medium-long chain triglyceride, adopts neutral alumina static adsorption and dynamic column chromatography coupling process, can greatly improve the purity of MLnM type triglyceride, far higher than other adsorbents, the whole process step is clear, including raw material preparation, static adsorption, dynamic chromatography and other links, operation process is easy to standardization and scale production, without relying on molecular distillation, supercritical CO2 Extraction and other complex equipment.

Description

Technical Field

[0001] This invention relates to the field of separation and purification technology, and in particular to a highly efficient method for the separation and purification of MLnM type medium- and long-chain triglycerides. Background Technology

[0002] Alpha-linolenic acid (ALA, C18:3ω-3), an essential fatty acid for the human body, is a key precursor in the synthesis of EPA / DHA and plays a vital role in preventing and treating cardiovascular diseases, inflammation, and metabolic syndrome. Its efficient delivery relies on specific molecular carriers. MLnM-type medium- and long-chain triglycerides (sn-1, 3-position medium-chain fatty acid M (C8-C12), sn-2-position α-linolenic acid Ln) are functional structural lipids that provide rapid energy and essential fatty acids. The sn-2-position ALA is hydrolyzed by pancreatic lipase into 2-monoglycerate, which is then absorbed intact in micelles, significantly improving bioavailability. The sn-1, 3-position MCFA is directly supplied to the liver via the portal vein for rapid energy delivery, reducing fat accumulation. However, the ALA in plant oils is mostly of the LnLL (L: long-chain fatty acid) type, resulting in low absorption rates. Even if the MLnM type is constructed, the product has a low content of the target MLnM type, mixed byproducts (free fatty acids (FFA), mono / diglycerides (MAG / DAG)) and unreacted substances, and there is an urgent need for efficient separation and purification technology to achieve industrialization.

[0003] Methods for separating medium- and long-chain triglycerides include molecular distillation, column chromatography, supercritical CO2 extraction, and solvent crystallization. Molecular distillation suffers from drawbacks such as high distillation temperatures, high equipment costs, and stringent parameter requirements. Supercritical CO2 extraction equipment is extremely expensive, with high-pressure systems costing 3-5 times more than molecular distillation equipment. ALA exhibits a dramatic increase in viscosity at low temperatures, becoming embedded in saturated TAG crystals. Furthermore, trace amounts of water in the oil form ice crystals at -20°C, disrupting the crystallization interface and thus reducing separation efficiency. Summary of the Invention

[0004] The purpose of this invention is to provide a highly efficient method for separating and purifying MLnM-type medium- and long-chain triglycerides. The method employs a coupled process of neutral alumina static adsorption and dynamic column chromatography, which can significantly improve the purity of MLnM-type triglycerides, far exceeding that of other adsorbents. The entire process is clearly defined, including raw material preparation, static adsorption, and dynamic chromatography. The operation process is easy to standardize and scale up for production, without relying on complex equipment such as molecular distillation and supercritical CO2 extraction.

[0005] To achieve the above objectives, this invention provides a highly efficient method for the separation and purification of MLnM-type medium- and long-chain triglycerides, comprising the following steps:

[0006] S1. Preparation of crude product;

[0007] S2. Mix the crude product with the adsorbent and perform static adsorption.

[0008] S3. The statically adsorbed product is eluted by a chromatography column, and the MLnM type triglycerides are separated by dynamic chromatography.

[0009] Preferably, the specific operation of S1 is as follows: using perilla seed oil and caprylic acid as substrates, immobilized lipase is added, and the mixture is shaken in an air bath constant temperature shaker to prepare a crude product containing MLnM type triglycerides through enzymatic acid hydrolysis.

[0010] Preferably, in S1, the molar ratio of perilla seed oil to caprylic acid is 1:2-4, the reaction temperature is 30-60℃, the shaking time is 5-8h, and the amount of enzyme added is 5-10wt% of the substrate.

[0011] Preferably, in S2, the amount of adsorbent is 5-15 wt% of the amount of raw material, the static adsorption temperature is 50-100℃, and the static adsorption time is 90-120 min.

[0012] Preferably, in S2, the adsorbent is neutral alumina.

[0013] Preferably, in S3, the column chromatography elution system is a petroleum ether / anhydrous diethyl ether system, with a volume ratio of petroleum ether to anhydrous diethyl ether of 1-15:1 and a flow rate of 1-2 mL / min.

[0014] Preferably, in S3, the column chromatography stationary phase is neutral alumina.

[0015] Therefore, the present invention employs the above-mentioned efficient separation and purification method for MLnM type medium- and long-chain triglycerides, using a coupled process of neutral alumina static adsorption and dynamic column chromatography, which can significantly improve the purity of MLnM type triglycerides, far exceeding that of other adsorbents. The entire process is clear, including raw material preparation, static adsorption, dynamic chromatography, and other steps. The operation process is easy to standardize and scale up for production, without relying on complex equipment such as molecular distillation and supercritical CO2 extraction.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This is a process flow diagram of Example 1 of the efficient separation and purification method for MLnM type medium- and long-chain triglycerides of the present invention;

[0018] Figure 2 This is the high performance liquid chromatography-mass spectrometry detection result of the elution fraction of Example 1 of the efficient separation and purification method for MLnM type medium- and long-chain triglycerides of the present invention;

[0019] Figure 3This is a diagram showing the separation effect of different adsorbents on the crude product in Example 1 and Comparative Examples 1-7 of the present invention, which describes an efficient separation and purification method for MLnM type medium- and long-chain triglycerides. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0023] The specific operating steps for dynamic adsorption in column chromatography are as follows:

[0024] (1) Wet packing

[0025] a. Installation location

[0026] First, fix the chromatographic column on the iron stand. Place a clean conical flask under the column. Use a glass rod to push a small amount of absorbent cotton into the narrow part at the bottom of the column. Add the prepared quartz sand into the column (about 5 mm). Pour a low-polarity solvent into the column to a height of about 1 / 4 of the column body. Gently tap the column body with the rubber bulb to expel the air from the quartz sand and absorbent cotton.

[0027] b. Wet packing

[0028] The pretreated stationary phase was placed in an Erlenmeyer flask, and soaked in solvent (petroleum ether, which has low polarity). The mixture swelled and was stirred into a paste. This paste was then poured into the chromatographic column using a solid feed funnel. The Erlenmeyer flask was then washed with a small amount of solvent to transfer the remaining stationary phase to the column. Finally, a small amount of solvent was used to flush any remaining adsorbent from the column wall into the column. After the transfer was complete, the stopcock at the bottom of the column was opened, and the column was tapped repeatedly or pressure was applied to allow the adsorbent to settle, ensuring that the solid adsorbent in the column was tightly packed and that the solvent level in the column was slightly higher than the adsorbent surface.

[0029] (2) Wet sampling

[0030] Using a spatula, gently cover the stationary phase surface with a layer of quartz sand, approximately 5 mm thick, along the column sidewall. Open the stopcock and close it when the solvent level drops to the quartz sand surface. Dissolve the deacidified structural lipid in a small amount of solvent, and gently add the sample solution dropwise onto the quartz sand using a long dropper. Open the stopcock until the solvent level drops to the quartz sand surface, then close it. Wash the sample flask with a small amount of eluent, add the liquid to the column, and wash away the sample from the sidewall. Open the stopcock until the solvent level drops to the quartz sand surface, then close it. Repeat the above steps until all crude product is adsorbed onto the stationary phase.

[0031] (3) Rinsing and collecting

[0032] After the sample loading is completed, add eluent along the side wall of the column, install the solvent bulb, and fix the column and solvent bulb with clamps. Then, add a large amount of eluent to the solvent bulb, receive it in a test tube, open the stopcock, apply pressure and rinse, and control the flow rate.

[0033] Pretreatment of silica gel and alumina: Spread commercially available silica gel and alumina separately on an iron tray and place them in a 120℃ oven for 3 hours to activate. After activation, remove them and place them in a drying oven.

[0034] Silver nitrate-silica gel pretreatment: Take 100g of silica gel (200-300 mesh), add 200mL of 96% (v / v) ethanol solution, stir for 10min to form a homogeneous slurry. Dissolve 10g of silver nitrate in 35mL of 70% (v / v) ethanol solution, add it dropwise to the silica gel slurry, and continue stirring for 10min. Transfer the mixture to a rotary evaporator and rotary evaporate the ethanol under vacuum at 60℃. Heat at 120℃ overnight to activate the silver-impregnated silica gel.

[0035] Macroporous resin pretreatment: Newly purchased resin contains some uncrosslinked monomer molecules and impurities, so it needs to be treated with acids and alkalis to remove these impurities before use. The specific steps are as follows: Accurately weigh 40g of resin, soak it in an appropriate amount of 95% ethanol for 24 hours, then filter the liquid and wash the resin with deionized water until the ethanol smell completely disappears; then soak it in 5% HCl for 4 hours, filter the liquid, and wash the resin with deionized water until the final filtrate is neutral (pH test paper); then soak it in 2% NaOH solution for 4 hours, filter the liquid, and wash the resin with deionized water until the final filtrate is neutral (pH=7); finally, soak it in ethanol, replacing the ethanol as needed, filter the resin dry, and store it in a 4℃ refrigerator for later use.

[0036] Example 1

[0037] This invention provides a highly efficient method for the separation and purification of MLnM type medium- and long-chain triglycerides, comprising the following steps:

[0038] S1. Preparation of crude product: Perilla seed oil and caprylic acid were used as substrates at a molar ratio of 1:3. 8 wt% of immobilized lipase was added to the substrates. The crude product containing MLnM type triglycerides was prepared by enzymatic acid hydrolysis. The mixture was shaken in an air bath constant temperature shaker at 45°C for 6 hours. The immobilized lipase was provided by Qingdao Weilan Biotechnology Co., Ltd.

[0039] S2. Mix the crude product with neutral alumina (10 wt% of the crude product mass) and slowly stir in a 70°C magnetically stirred water bath for 120 min to obtain the static adsorption product.

[0040] S3. Pass the statically adsorbed product through a neutral alumina chromatography column. The elution system is: petroleum ether / anhydrous diethyl ether (5:1, v / v), the flow rate is 1.5 mL / min, the stationary phase is 40 g, and the sample loading amount is 4 g.

[0041] Example 2

[0042] The difference between Example 2 and Example 1 is that in Example 2, the ratio of petroleum ether to anhydrous diethyl ether is 1:1, while all other conditions are the same.

[0043] Example 3

[0044] The difference between Example 3 and Example 1 is that in Example 3, the ratio of petroleum ether to anhydrous diethyl ether is 15:1, while all other conditions are the same.

[0045] Comparative Example 1

[0046] The difference between Comparative Example 1 and Example 1 is that the adsorbent used in Comparative Example 1 is silica gel, while all other conditions are the same.

[0047] Comparative Example 2

[0048] The difference between Comparative Example 2 and Example 1 is that the adsorbent used in Comparative Example 2 is silver nitrate-silica gel, while all other conditions are the same.

[0049] Comparative Example 3

[0050] The difference between Comparative Example 3 and Example 1 is that the adsorbent used in Comparative Example 3 is a polar macroporous resin, while all other conditions are the same.

[0051] Comparative Example 4

[0052] The difference between Comparative Example 4 and Example 1 is that the adsorbent used in Comparative Example 4 is a medium polar macroporous resin, while all other conditions are the same.

[0053] Comparative Example 5

[0054] The difference between Comparative Example 5 and Example 1 is that the adsorbent used in Comparative Example 5 is a weakly polar macroporous resin, while all other conditions are the same.

[0055] Comparative Example 6

[0056] The difference between Comparative Example 6 and Example 1 is that the adsorbent used in Comparative Example 6 is a non-polar macroporous resin, while all other conditions are the same.

[0057] Comparative Example 7

[0058] The difference between Comparative Example 7 and Example 1 is that the stationary phase used in Comparative Example 7 is silica gel, while all other conditions are the same.

[0059] Comparative Example 8

[0060] The difference between Comparative Example 8 and Example 1 is that the stationary phase used in Comparative Example 8 is silver nitrate-silica gel, while all other conditions are the same.

[0061] Comparative Example 9

[0062] The difference between Comparative Example 9 and Example 1 is that the stationary phase used in Comparative Example 9 is a polar macroporous resin, while all other conditions are the same.

[0063] Comparative Example 10

[0064] The difference between Comparative Example 10 and Example 1 is that the stationary phase used in Comparative Example 10 is a medium polar macroporous resin, while all other conditions are the same.

[0065] Comparative Example 11

[0066] The difference between Comparative Example 11 and Example 1 is that the stationary phase used in Comparative Example 11 is a weakly polar macroporous resin, while all other conditions are the same.

[0067] Comparative Example 12

[0068] The difference between Comparative Example 12 and Example 1 is that the stationary phase used in Comparative Example 12 is a non-polar macroporous resin, while all other conditions are the same.

[0069] The crude product obtained in Example 1 was used to prepare a sample stock solution with a concentration of 5 mg / mL. This stock solution was then serially diluted with isopropanol solution to obtain a sample stock solution with a concentration of 50 μg / mL. One mL of this stock solution was pipetted into a sample vial, filtered through a 0.22 μm organic filter membrane, and then injected.

[0070] UPLC-MS / MS conditions: Mobile phase A was acetonitrile / methanol / water (19 / 19 / 2, v / v / v), mobile phase B was isopropanol (5 mmol / L ammonium acetate and 0.1% formic acid were added to both mobile phases A and B), column temperature was 35℃, injection volume was 1 μL, and elution conditions for the mobile phases are shown in Table 1.

[0071] Table 1 Elution conditions for ultra-high performance liquid chromatography-mass spectrometry

[0072]

[0073]

[0074] MS conditions: ESI ionization source, positive ion mode, capillary voltage 3.1 kV, cone voltage 40.0 V, ion source temperature 120 °C, desolventizing temperature 450 °C, cone gas flow rate 80.0 L / h, desolventizing gas flow rate 600.0 L / h, scanning mode MSE, first-order collision voltage 6 V, second-order collision voltage 20-40 V, scan interval 0.3 s, molecular weight scan range 100-1200 m / z, acquisition time 15 min. Argon was used as the collision gas.

[0075] The elution fraction results are as follows Figure 2 As shown, combined with Figure 2 Chromatographic analysis of the eluted fractions showed that the column exhibited excellent separation performance for TAG components after neutral alumina coupled adsorption treatment. The chromatographic peak heights of the byproducts MCT (retention time approximately 2 min) and LCT (retention time approximately 12 min) were relatively low, indicating that they were effectively removed. MLCT-related components formed a significant main peak group in the 6–10 min range. The target product MLnM showed multi-stage elution peaks in the 6–10 min range, with its total peak area accounting for 68.48% of the total TAG amount. Together with MLM, it constituted 90.62% of the main components. The total MLCT accounted for as high as 90%–98% of the TAG, indicating that the selective retention of triglycerides with different carbon chain lengths by the polar adsorption medium effectively separated medium-chain (MCT), long-chain (LCT), and target medium- and long-chain components.

[0076] Comparing Examples 1-3, Table 2 shows that when the eluent ratio is 5:1 (v / v), the separation degree reaches 1.5, which is the highest among all ratios. This indicates that under this ratio, MLnM type medium-long chain triglycerides can achieve relatively good separation effect from other substances.

[0077] When the eluent ratio is 15:1 (v / v), the separation degree is 1.2, which is the lowest among all ratios, indicating that the separation effect of this ratio on MLnM type medium and long chain triglycerides is relatively poor.

[0078] Table 2. Separation effect of thin-layer chromatography with different eluent ratios

[0079] 1:1 1.3 5:1 1.5 15:1 1.2

[0080] To investigate the separation effect of the adsorbent on the crude product by comparing Example 1 and Comparative Examples 1-6, the crude products of Example 1 and Comparative Examples 1-6 were mixed with the activated adsorbent and placed in a 70℃ water bath with slow stirring for 2 hours. After removal, the mixture was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected as the static adsorption product. The results are shown in Table 3 and... Figure 3 As shown.

[0081] Table 37 shows the separation effects of 37 adsorbents on the static adsorption of crude products.

[0082]

[0083]

[0084] From Table 3 and Figure 3 It can be seen that alumina is significantly better than other adsorbents in terms of both enrichment and impurity removal of MLnM during static adsorption, making it the optimal choice. Weakly polar macroporous resins are the next best, while polar and moderately polar macroporous resins are the least effective.

[0085] To investigate the separation effect of the stationary phase on the crude product, 4g of the static adsorption product of Example 1 and Comparative Examples 7-12 and 25g of stationary phase were weighed. The column was packed using a wet method with petroleum ether / anhydrous diethyl ether at a ratio of 5:1 as the eluent. Isocratic elution was performed at a flow rate of 1.5mL / min. Components with different polarities were collected, and the results are shown in Table 4.

[0086] Table 47 shows the separation effect of stationary phases on static adsorption products of alumina.

[0087]

[0088]

[0089] As shown in Table 4, the enrichment rates of MLnM by the seven fixed phases ranged from 40% to 70%, and the proportion of MLnM containing essential fatty acids to the total MLM ranged from 68% to 91%. Among them, the static-dynamic coupling adsorption method of neutral alumina was the best separation method for crude MLnM products.

[0090] Therefore, the present invention employs the above-mentioned efficient separation and purification method for MLnM type medium- and long-chain triglycerides, using a coupled process of neutral alumina static adsorption and dynamic column chromatography, which can significantly improve the purity of MLnM type triglycerides, far exceeding that of other adsorbents. The entire process is clear, including raw material preparation, static adsorption, dynamic chromatography, and other steps. The operation process is easy to standardize and scale up for production, without relying on complex equipment such as molecular distillation and supercritical CO2 extraction.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A highly efficient method for the separation and purification of MLnM type medium- and long-chain triglycerides, characterized in that: Includes the following steps: S1. Preparation of crude product; The specific operation of S1 is as follows: using perilla seed oil and caprylic acid as substrates, immobilized lipase is added, and the mixture is shaken in an air bath constant temperature shaker to prepare crude product containing MLnM type triglycerides through enzymatic acid hydrolysis. S2. Mix the crude product with the adsorbent and perform static adsorption. In S2, the amount of adsorbent used is 5-15 wt% of the crude product, the static adsorption temperature is 50-100℃, and the static adsorption time is 90-120 min. In S2, the adsorbent is neutral alumina; S3. The static adsorption product is eluted by column chromatography, and the MLnM type triglycerides are separated by dynamic chromatography. In S3, the column chromatography elution system is a petroleum ether / anhydrous diethyl ether system, with a volume ratio of petroleum ether to anhydrous diethyl ether of 1-15:1 and a flow rate of 1-2 mL / min. In S3, the stationary phase for column chromatography is neutral alumina.

2. The efficient separation and purification method for MLnM type medium-long chain triglycerides according to claim 1, characterized in that: In S1, the molar ratio of perilla seed oil to caprylic acid is 1:2-4, the reaction temperature is 30-60℃, the shaking time is 5-8h, and the amount of enzyme added is 5-10wt% of the substrate.

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